Methods involving the kinetic analysis of in vitro nucleic acid amplification have become important tools for quantifying analyte polynucleotides. In these procedures, sometimes referred to as “real-time” amplification procedures, the amount of amplicon present in a nucleic acid amplification reaction mixture is monitored as a function of time over the course of the amplification procedure. Fully automated real-time nucleic acid assays require machine executable algorithms capable of analyzing the time-dependent data acquired during the reaction. In this regard, there is a requirement for data processing algorithms that accurately output an amount or concentration of a nucleic acid that would give rise to an observed amplification result.
Difficulties associated with quantifying the absolute amount of a specific nucleic acid target have been appreciated in the patent literature. These difficulties have been attributed to the exponential nature of the amplification process, and the fact that small differences in any of the variables which control the reaction rate, including the length and nucleotide sequence of the primer pairs, can lead to dramatic differences in amplicon yield. Wang et al., in U.S. Pat. No. 5,219,727 described the use of an internal standard that amplified using the same primers that amplified the analyte polynucleotide, and addressed the fact that use of an unrelated cDNA as a standard necessitated a second set of oligonucleotide primers unrelated to the specific target nucleic acid being quantified. According to Wang et al., analyses which use two sets of unrelated primers can only provide a relative comparison of two independent amplification reactions rather than an absolute measure of a nucleic acid target concentration. Others have followed this teaching and employed internal standards that resemble the target of interest by having similar sequences, and by amplifying with a common pair of primers (see published U.S. patent application Ser. No. 10/230,489). Still others have described quantitative methods that rely on determining the efficiency of amplification (see published European Patent Application EP 1138784). Yet another approach has involved determining amplification ratios for control and target sequences (see U.S. Pat. No. 6,066,458).
Yet other approaches have been used both to improve the quality of quantitative results obtained using real-time nucleic acid amplification, and to simplify assay procedures. One approach involves the use of factory-produced calibration curves that can be loaded into a data processing component of an automated analyzer. The use of such calibration curves, sometimes referred to as “stored master curves,” advantageously reduces the number of calibration standard reactions that must be supported by manufacturing, and reduces the number of reactions that must be performed by end users. Since the use of a stored master curve makes it unnecessary to create a complete calibration curve every time an assay is performed, the requirement for batch testing that would otherwise employ a contemporaneously generated local calibration plot is eliminated.
The invention described herein provides a mechanism for adjusting stored master calibration curves using results obtained by running only a very small number of calibration standard reactions.